應用萊吉氏細胞篩選中草藥之促性腺有效成分(2/2)

The purpose of this study was
firstly to isolate purified mice Leydig cells
and use enzyme immunoassay (EIA) for
testosterone production. Selected Chinese
medicinal herbs extracted by hot water
extraction procedure were co-cultured with
the isolated cells and their effect on
testosterone secretion vis a vis basal and
stimulants were investigated.
Testis were obtained from mature
ICR mouse of 6-8 weeks age, decapsulated
and finely minced before dissociating with
collagenase. The digested tissue is then
sieved and Percoll discontinuous gradients
method applied to separate the Leydig cells.
The harvested Leydig cells were than
incubated in M199 in a 48 well culture plate,
different doses of Chinese medicinal herbs
extracts, various stimulants and signal
transductor inhibitors were added to the
Leydig cells culture system. The medium
was than collected at different time periods
and assayed using EIA technique. The
results and findings of our present work can
be summarized as follows:
1. Investigating for 10, 20, 30, 40 and 50
% Percoll discontinuous gradients and
using LH stimulation and 3-HSD
staining it was revealed that 50%
Percoll gradients has better results. The
Leydig cells when stimulated (LH,
cAMP) had good testosterone
production both at dose and time
response indicating the successful
establishment of mouse Leydig cells in
vitro cell culture system.
2. Tribulus terrestris L. hot water extracts
(H13) stimulated testosterone secretion
of mouse Leydig cells, in 100 µg/ml
dose almost double to that of basal. It
also showed time course (24 and 48
hour) and dose response (1, 10 and
100µg/ml). It also revealed that the H13
mediated stimulation via PKA and PKC
activation leading to protein
transcription and translation.
3. Cinnamomum cassia Presl extracted by
hot water extraction (10 and 100 µg/ml)
showed decrease in testosterone
production over the basal and LH
stimulated secretion of mouse Leydig
cells in 4 and 24 hour time course. From
our data by addition of signal
transduction agents, it was suggested
that Cinnamomum cassia Presl decrease
LH stimulated testosterone secretion
function without acting on the
steroidogensis pathway.